Technical advances in the sequencing of DNA have led to the sequencing of numerous genomes from simple bacteria to complex eukaryotic genomes, including humans. Great advances have also been realized in the ever expanding field of proteomics, which is involved with the identification, expression, structure, and function of proteins produced by the genome.
The past decade has seen rapid development in the field with the expansion in the use of microarrays to analyze protein expression and abundance. The clinical area of proteomic research has been greatly advanced by the use of two-dimensional gel electrophoresis combined with mass spectroscopic sequencing to analyze protein expression in pathogenic microorganisms and to identify protein targets for cancer therapy.
Functional proteomics' ultimate goal is to determine what each protein in an organism does, how it does it, and examine the myriad of conditions that influence its activity. To accomplish this task, proteins must be either purified from their natural environment or produced using recombinant DNA technology—the only practical method to achieve this goal.
During the past several years methods have been developed for the cloning of PCR generated open reading frame (ORF) libraries in bacterial entry vectors that could be transferred to a variety of expression vectors (destination, vectors) using site specific recombination technology that maintains the proper reading frame and gene orientation. The incorporation of affinity tags is usually required during plasmid construction to facilitate purification and concentration of the target proteins. Scoring of positive results is initially performed on standard Coomassie stained protein gels or Western blots after high-throughput purification using robotic platforms.
Numerous expression systems could be used for high-throughput, robotic production and purification of recombinant proteins using a variety of prokaryotic and eukaryotic inducible vectors and hosts. All of these systems rely on tight regulation of transcription during logarithmic phase of growth followed by chemical, induction to initiate protein production. There is a great need for the development of expression systems that are more adaptable for large-scale culture and also suitable for use as a high-throughput vector.